RESEARCH ARTICLE

Combination of Cefditoren and N-acetyl-L-Cysteine Shows a

Synergistic Effect against Multidrug-Resistant Streptococcus
pneumoniae Biofilms
Mirella Llamosí,a Julio Sempere,a,b Pilar Coronel,c Mercedes Gimeno,c Jose Yuste,a,b Mirian Domenecha,b,d

a Spanish Pneumococcal Reference Laboratory, National Center for Microbiology, Instituto de Salud Carlos III, Madrid, Spain
b CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain
c Scientific Department, Meiji Pharma Spain, Madrid, Spain
d Department of Genetics, Physiology, and Microbiology, Faculty of Biology, Complutense University of Madrid, Madrid, Spain

Mirella Llamosí and Julio Sempere contributed equally to this work. Author order was determined by seniority.

ABSTRACT Biofilm formation by Streptococcus pneumoniae is associated with coloniza-

tion of the upper respiratory tract, including the carrier state, and with chronic respiratory
infections in patients suffering from chronic obstructive pulmonary disease (COPD). The
use of antibiotics alone to treat recalcitrant infections caused by biofilms is insufficient in
many cases, requiring novel strategies based on a combination of antibiotics with other
agents, including antibodies, enzybiotics, and antioxidants. In this work, we demonstrate
that the third-generation oral cephalosporin cefditoren (CDN) and the antioxidant N-ace-
tyl-L-cysteine (NAC) are synergistic against pneumococcal biofilms. Additionally, the com-
bination of CDN and NAC resulted in the inhibition of bacterial growth (planktonic and
biofilm cells) and destruction of the biofilm biomass. This marked antimicrobial effect was
also observed in terms of viability in both inhibition (prevention) and disaggregation
(treatment) assays. Moreover, the use of CDN and NAC reduced bacterial adhesion to
human lung epithelial cells, confirming that this strategy of combining these two com-
pounds is effective against resistant pneumococcal strains colonizing the lung epithelium.
Finally, administration of CDN and NAC in mice suffering acute pneumococcal pneumo-
nia caused by a multidrug-resistant strain was effective in clearing the bacteria from the
respiratory tract in comparison to treatment with either compound alone. Overall, these
results demonstrate that the combination of oral cephalosporins and antioxidants, such
as CDN and NAC, respectively, is a promising strategy against respiratory biofilms caused
by S. pneumoniae. Editor Fikri Y. Avci, Emory University School of
Medicine
IMPORTANCE Streptococcus pneumoniae is one of the deadliest bacterial pathogens,
Copyright © 2022 Llamosí et al. This is an
accounting for up to 2 million deaths annually prior to severe acute respiratory syndrome open-access article distributed under the terms
coronavirus 2 (SARS-CoV-2). Vaccines have decreased the burden of diseases produced by of the Creative Commons Attribution 4.0
International license.
S. pneumoniae, but the rise of antibiotic-resistant strains and nonvaccine serotypes is worri-
Address correspondence to Jose Yuste,
some. Pneumococcal biofilms are associated with chronic respiratory infections, and treat- jyuste@isciii.es, or Mirian Domenech,
ment is challenging, making the search for new antibiofilm therapies a priority as biofilms miridome@ucm.es.
become resistant to traditional antibiotics. In this work, we used the combination of an an- The authors declare a conflict of interest. J.Y.
has received grants from MSD-USA (MISP Call),
tibiotic (CDN) and an antioxidant (NAC) to treat the pneumococcal biofilms of relevant
and Pfizer that are not related to this work. J.Y.
clinical isolates. We demonstrated a synergy between CDN and NAC that inhibited and has participated in advisory boards organized
treated pneumococcal biofilms, impaired pneumococcal adherence to the lung epithelium, by MSD, and Pfizer. M.G. and P.C. are members
of the Scientific Department of Meiji Pharma
and treated pneumonia in a mouse pneumonia model. We propose the widely used Spain. The other authors declare no competing
cephalosporin CDN and the repurposed drug NAC as a new antibiofilm therapy against interests.
S. pneumoniae biofilms, including those formed by antibiotic-resistant clinical isolates. Received 7 September 2022
Accepted 14 November 2022
KEYWORDS biofilm, pneumococcus, serotype 19A, antioxidants, N-acetyl-L-cysteine, Published 29 November 2022
cefditoren

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A ccording to recent data from the World Health Organization (1), respiratory tract
diseases remain one of the main causes of death in the world in 2019. Lower respi-
ratory tract infections are the world’s most deadly communicable disease and are the
fourth leading cause of death without considering severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) pandemic data, whereas chronic obstructive pulmonary
disease (COPD) claimed another 3 million lives worldwide in 2019 (1). In addition, acute
respiratory infections are the main cause of mortality in children under 5 years, with
Streptococcus pneumoniae (pneumococcus) being the principal cause of severe pneu-
monia in both developing and developed countries. Acquisition of new strains of
S. pneumoniae is associated with acute exacerbations of COPD (AECOPD) in current
and former smokers (2, 3), with this bacterial pathogen being one of the most frequent
in COPD patients, although many other chronic bacterial infections are also produced
by communities of diverse species (4).
Notably, 65 to 80% of chronic bacterial infections are caused by microbes growing in
biofilms (5). The presence of biofilms is well documented in otitis media and persistent
pneumonia, which are difficult diseases to treat with antibiotics and are often chronic and
recurrent. Most of the information available so far is related to S. pneumoniae (6). The in-
herent tolerance of these communities to antibiotic therapy and the host immune system
is well known (7–10). Biofilms may be established on medical devices, such as indwelling
intravenous catheters or endotracheal tubes, when local defenses are impaired (11). The
ability of respiratory pathogens to persist in the nasopharynx and cause infection under
the appropriate conditions is associated with the ability to form biofilms on the mucosal
epithelium (12). Current strategies for inhibition of biofilm formation or disrupting estab-
lished biofilms represent an exciting new approach to the treatment of chronic infectious
diseases. The application of these strategies to respiratory tract infections also offers a bet-
ter understanding of the significance of mucosal biofilm in the pathogenesis of these
conditions.
S. pneumoniae carriage is a prerequisite to the later invasion of sterile sites, leading
to what is known as invasive pneumococcal disease (IPD). Indeed, the pneumococcus
is responsible for episodes of sepsis, bacteremic pneumonia, and meningitis, mainly in
children, the elderly, and immunocompromised patients (13). In addition, pneumococci
are the main bacterial cause of noninvasive diseases such as nonbacteremic pneumonia,
acute otitis media, sinusitis, and conjunctivitis (14). One of the major threats in pneumo-
coccal infection is the emergence of nonvaccine serotypes, causing IPD associated with
antibiotic resistance (15, 16).
Numerous strategies are being actively pursued to inhibit biofilm formation or erad-
icate established biofilms of respiratory pathogens (17, 18). Bacteria forming a biofilm
can be up to 1,000 times more resistant to antimicrobial agents than planktonic cells
(19), and in this sense, new antibiofilm therapies are necessary from the prophylactic
and therapeutic perspectives as alternative or complementary options to common
antibiotics. The mucolytic agent N-acetyl-L-cysteine (NAC), a precursor of the antioxi-
dant glutathione, has been investigated for its antimicrobial activity in the inhibition of
biofilm formation and in the clearance of developed biofilms. The ability of NAC to
interfere with bacterial growth and biofilm formation was first demonstrated against
Staphylococcus epidermidis, confirming a concentration-dependent effect (20). Since
then, many other studies have demonstrated the efficacy of NAC in reducing biofilm
formation in a wide range of microorganisms (including Gram-negative and Gram-pos-
itive bacteria, as well as yeasts), demonstrating its ability to impair matrix architecture
and promote the disruption of biofilms (6, 21–23). In addition, the combination of anti-
biotics with other alternative therapies is a promising and effective strategy against
bacterial biofilms (24, 25).
In this study, we used cefditoren (CDN), a third-generation oral cephalosporin, which has
demonstrated bactericidal activity against many Gram-negative and Gram-positive bacterial
pathogens. Previous publications have shown that CDN has good in vitro activity against the
two major bacterial respiratory pathogens, S. pneumoniae and Haemophilus influenzae (26).

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The main objective of this work was to explore the ability of cefditoren alone and in combina-
tion with NAC to inhibit (prevent) and degrade (treat) biofilms produced by S. pneumoniae
serotype 19A strains with different antibiotic susceptibility patterns.

RESULTS
Cefditoren showed a marked antimicrobial activity against S. pneumoniae
(planktonic and biofilm). To explore the ability of CDN to prevent S. pneumoniae
growth and biofilm formation, we used clinical strains of serotype 19A with different
susceptibilities to CDN (3064/19, susceptible; 1171/19, intermediate; and 3152/19,
resistant). To compare the results obtained with CDN, we used the antibiotic cefixime
(CFM), which is also a third-generation oral cephalosporin. First, we tested the bacterio-
lytic activity of CDN and CFM separately against the bacterial growth of the different
clinical isolates (Fig. 1A to C). The results clearly proved that CDN displays a strong bac-
teriolytic effect, even against the multidrug-resistant (MDR) strain 3152/19 (Fig. 1C),
whereas when CFM was used, the viability of the culture was recovered after 8 h of
incubation with the antibiotic, even for the susceptible strain, 3064/19 (Fig. 1A). These
results confirm that CDN has stronger antimicrobial activity than CFM.
In addition, we evaluated the effect of different concentrations of CDN and CFM
against bacterial growth (including planktonic and biofilm) and against established
biofilms (biofilm biomass) of the different S. pneumoniae strains growing in microwell
plates (Fig. 2). We performed an inhibition (prevention) assay, where different concen-
trations of CDN or CFM (0.2, 0.4, 0.8, and 1 m g/mL) were added at the beginning of the
incubation along with the inoculum (Fig. 2). For this purpose, we measured the total
growth and biofilm biomass after 5 h of incubation (Fig. 2). Treatment with CDN was
very effective at inhibiting the total bacterial growth for all the clinical isolates (Fig. 2A,
C, and E). For the susceptible strain (3064/19), exposure to the lowest dose of CDN
(0.2 m g/mL) was enough to show an almost complete eradication of pneumococcal
cells, whereas for CFM, concentrations up to four times higher were necessary to
observe a similar effect (0.8 m g/mL) (Fig. 2A). For the intermediate (1171/19) and resist-
ant (3152/19) strains, higher doses of CFM than CDN were required to reduce the total
growth of the pneumococcal cells (Fig. 2C and E). In terms of prevention of pneumo-
coccal biofilm biomass (Fig. 2B, D, and F), an almost complete reduction in the biofilm
of the susceptible strain (3064/19) was observed with the lowest concentration of CDN
(0.2 m g/mL), producing a 96% reduction of the pneumococcal biofilm (Fig. 2B). However,
for CFM, a concentration up to 5 times higher (1 m g/mL) was necessary to achieve a similar
reduction of the pneumococcal biofilm (Fig. 2B). In the pneumococcal strain with interme-
diate resistance to CDN (1171/19), the use of CDN as a preventive measure showed effec-
tiveness in inhibiting the biofilm formation at all the doses evaluated, leading to a 90%
reduction of the biofilm with a dose of 1 m g/mL (Fig. 2D). However, for CFM, higher doses
were necessary to show a certain degree of reduction, confirming that CFM had a weaker
ability to prevent biofilms by nonsusceptible strains than CDN (Fig. 2D). In the case of the
pneumococcal strain resistant to CDN and CFM, the inhibitory effect of both antibiotics
against the pneumococcal biofilm was similar, showing bactericidal activity with increasing
doses (Fig. 2F).
Effects of the combination of CDN and NAC on the growth, formation, and dis-
aggregation of S. pneumoniae biofilms. We characterized the combined effect of
cephalosporins and NAC against pneumococcal biofilms of the different strains of
S. pneumoniae (Fig. 3). First, we evaluated the impact of adding NAC, CDN, and CFM
alone and a combination of NAC with each cephalosporin on the growth curves of the
MDR strain 3152/19 (Fig. 3A and B). Our results confirmed that the combination of
1.5 mg/mL of NAC and 0.4 m g/mL of CDN was more effective than individual treat-
ments at inhibiting the growth of this resistant pneumococcal strain (Fig. 3A).
However, the combination of NAC with CFM at the same concentrations had no effect
(Fig. 3B). Thereafter, we only tested NAC and CDN.
We also analyzed the combined effect of NAC and CDN on the total growth and
biofilm of the different serotype 19A strains in a prevention/inhibition assay (Fig. 3C

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FIG 1 Inhibition of S. pneumoniae growth curves with third-generation cephalosporins. CDN (0.4 m g/mL)
or CFM (0.4 m g/mL) were added at the beginning with the inoculum, and the cultures were incubated
for 24 h at 37°C. (A) Growth curve of the susceptible strain 3064/19. (B) Growth curve of the intermediate
strain 1171/19. (C) Growth curve of the MDR strain 3152/19. C, control.

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FIG 2 Inhibition (prevention) of S. pneumoniae biofilms with the third-generation cephalosporins CDN and CFM. Different concentrations
of CFM or CDN (0 to 1 m g/mL) were added at the beginning with the inoculum, and the biofilms were incubated for 5 h at 37°C. We
used the susceptible strain 3064/19 (A and B), the intermediate strain 1171/19 (C and D), and the MDR strain 3152/19 (E and F). Black
bars represent the total growth (planktonic plus biofilm) (A, C, and E), and the white/colored bars represent the biofilm biomass (B, D,
and F). Data represent the average of three experiments. Standard deviation bars are shown, and asterisks mark results that are
statistically significant (two-tailed Student’s t test: *, P , 0.05; **, P , 0.01; ***, P , 0.001).

and D). Treatment with CDN was more effective than treatment with NAC at prevent-
ing the formation of pneumococcal biofilms for all the investigated strains (Fig. 3C and
D). For the susceptible strain (3064/19), individual treatments with 2.5 mg/mL of NAC
or 0.4 to 0.8 m g/mL of CDN caused a dramatic decrease in the growth, and therefore,
additive or synergistic effects mediated by the combination of NAC with CDN were not
possible to distinguish from individual treatments (Fig. 3C). However, for the interme-
diate (1171/19) and resistant strains (3152/19), the combination of NAC and CDN
achieved greater effectiveness in reducing the total growth (Fig. 3C).
In terms of inhibition of the biofilm biomass (Fig. 3D), for the susceptible strain
(3064/19), the individual treatments were highly effective, as we observed for the total
growth, and we could not quantify the impact of the combination (Fig. 3D). However,

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FIG 3 Effect of the combination of NAC and CDN/CFM on the growth, formation, and disaggregation of
S. pneumoniae biofilms. (A and B) Effect of treatments on growth curves of the MDR strain S. pneumoniae 3152/19.
NAC (1.5 mg/mL), CDN (0.4 m g/mL), CFM (0.4 m g/mL), or a combination of antibiotic plus antioxidant was added at
the beginning with the inoculum, and the cultures were incubated for 24 h at 37°C. (C and D) Inhibition of
S. pneumoniae biofilms with different concentrations of NAC (2.5 mg/mL), CDN (0.4 or 0.8 m g/mL), or a combination
of NAC and CDN. Treatments were added at the beginning with the inoculum, and the biofilms were incubated for
5 h at 37°C. We used the susceptible strain 3064/19, the intermediate strain 1171/19 and the MDR strain 3152/19.
(E) Viable counts showing the inhibition (prevention) of MDR strain 3152/19 biofilm with CDN and NAC. NAC
(2.5 mg/mL), CDN (0.8 m g/mL), or a combination of NAC and CDN was added at the beginning with the inoculum,
and the biofilms were incubated for 5 h at 37°C. Viability was assessed at the end of the experiment and
normalized to the control. (F) Viable counts showing the disaggregation (treatment) of S. pneumoniae biofilms with
CDN and NAC. Biofilms were inoculated and incubated for 4 h at 37°C and then treated with CDN (2 m g/mL) or a
combination of NAC (2.5 mg/mL) and CDN for 1.5 h at 37°C. Viability was assessed at the end of the experiment
and normalized to CDN. Data represent the average of three experiments. Standard deviation bars are shown, and
asterisks mark results that are statistically significant (two-tailed Student’s t test: ***, P , 0.001).

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for the intermediate and resistant strains of serotype 19A, much higher antimicrobial
activity was observed when NAC and CDN were combined in comparison to the indi-
vidual treatments, confirming the potential effectiveness of this combination in the
prevention of pneumococcal biofilms (Fig. 3D). Based on these results, further studies
exploring the activity against pneumococcal biofilms were performed using the resist-
ant strain (3152/19) (Fig. 3E). Viability studies demonstrated that NAC and CDN had a
similar effect against pneumococcal cells within the biofilm, yielding the observation
that the combination of 2.5 mg/mL of NAC and 0.8 m g/mL of CDN reduced the biofilm
viability by up to 95%, indicating a stronger effect compared to the individual treat-
ments with CDN or NAC (Fig. 3E). Overall, these results confirm a potential synergistic
effect between NAC and CDN on the growth and formation of pneumococcal biofilms.
To set up the optimal conditions for treatment assays with established pneumococ-
cal biofilms, different amounts of CDN (0.5 to 6 m g/mL) were tested to evaluate the
ability of CDN to disaggregate established mature pneumococcal biofilms. For the
MDR strain (3152/19), the viability of the biofilm was reduced by 1 log at 0.5 m g/mL
and up to 2 logs at 2 m g/mL (data not shown). Based on these preliminary results, a
combination of 2.5 mg/mL of NAC with 2 m g/mL of CDN was used for the disaggrega-
tion assays against the three different pneumococcal strains of serotype 19A (Fig. 3F).
The combined treatment resulted in a 9% reduction in the CDN-susceptible strain
(3064/19), a 50% reduction in the CDN intermediate strain (1171/19), and a 30% reduc-
tion in the MDR strain (3052/19) in comparison with treatment with CDN alone
(Fig. 3F). Hence, the synergistic effect between NAC and CDN was demonstrated for all
the strains with reduced susceptibility to CDN.
Treatment with the combination of CDN and NAC impaired the attachment of
S. pneumoniae to the lung epithelium and was effective against pneumococcal
pneumonia. The antimicrobial effect of the combination of CDN and NAC against pneu-
mococcal adhesion in the lower respiratory tract was explored using human lung epithelial
cells. We studied the individual effect of both drugs and the combination by analyzing the
viable bacterial counts, and images of the infected cells were obtained using confocal laser
scanning microscopy (CLSM). For these assays, pneumococcal strains with reduced suscep-
tibility to CDN (strains 1171/19 and 3152/19) were used (Fig. 4 and 5). In the viability assay,
the percentage of adhesion compared to the control was determined, and we found that
exposure to 0.4 m g/mL of CDN was more effective than 2.5 mg/mL of NAC at reducing the
adhesion of both S. pneumoniae strains (Fig. 4A and 5A). These results agree with previous
findings shown above related to the prevention of biofilm assays (Fig. 3). Moreover, the
combination of CDN and NAC reduced the adhesion to a higher degree (P , 0.001) than
each compound individually, demonstrating again a synergistic effect (Fig. 4 and 5). In the
CLSM images, we also observed an increased effect of the combined treatment compared
to the individual treatments for both strains (Fig. 4B and 5B). These results indicate that
the combination of CDN and NAC displays a synergistic antimicrobial effect against pneu-
mococcal attachment to lung epithelial cells by clearing the adhesion of the bacteria to
the respiratory tract (Fig. 4B and 5B).
To evaluate the in vivo relevance of our findings, a pneumonia model of infection
using the MDR strain (3152/19) was studied in mice to mimic pneumococcal pneumonia.
For this purpose, mice anesthetized with isoflurane were infected via intranasal inocula-
tion with the infective strain resistant to CDN. After 24 h of infection, the mice were
treated with 1 dose every 12 h of humanized doses of NAC (5.7 mg/kg), CDN (4.3 mg/kg),
or the combination, as recommended by the manufacturer; the mice were sacrificed at
48 h postinfection to analyze the bacterial levels in the lungs. In Fig. 6, we show the bacte-
rial counts (log10 CFU per milliliter) recovered from the lungs of infected mice at 48 h post-
infection (after 24 h of treatment). We did not see significant differences in the groups
treated with NAC or CDN individually (Fig. 6), which is consistent with the adhesion assays
using human lung epithelial cells. However, treatment with the combination of CDN and
NAC significantly reduced the bacterial load in the lung compared to the individual treat-
ments, confirming that the CDN and NAC combination is synergistic against pneumococ-
cal pneumonia, even against a strain resistant to CDN (Fig. 6).

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FIG 4 Effect of the combination of NAC and CDN on adhesion of the intermediate strain S. pneumoniae 1171/19 to
the lung epithelium. A549 cells were prepared to a density of 105 cells per well, infected with strain 1171/19, and
incubated for 2 h at 37°C. The culture was then treated with NAC (2.5 mg/mL), CDN (0.4 m g/mL), or a combination
of CDN plus NAC and incubated for 1 h at 37°C. (A) Box and whisker plot showing viability. Viability was assessed at
the end of the experiment and normalized to the control. Data represent the average of three experiments.
Standard deviation bars are shown, and asterisks mark results that are statistically significant (two-tailed Student’s t
test: *, P , 0.05; ***, P , 0.001). (B) DNA was stained with Hoechst solution, the actin cytoskeleton was visualized
with rhodamine-phalloidin staining, and bacterial strain 1171/19 was fluorescently labeled with FAM-SE. Images
represent the xy from z-stacks at 0.5-m m intervals.

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FIG 5 Effect of the combination of NAC and CDN on adhesion of the MDR strain S. pneumoniae 3152/19 to the
lung epithelium. A549 cells were prepared to a density of 105 cells per well, infected with strain 3152/19, and
incubated for 2 h at 37°C. The culture was then treated with NAC (2.5 mg/mL), CDN (0.4 m g/mL), or a combination
of CDN plus NAC and incubated for 1 h at 37°C. (A) Box and whisker plot showing viability. Bars graph showing
viability was assessed at the end of the experiment and normalized to the control. Data represent the average of
three experiments. Standard deviation bars are shown, and asterisks mark results that are statistically significant
(two-tailed Student’s t test: *, P , 0.05; ***, P , 0.001). (B) DNA was stained with Hoechst solution, the actin
cytoskeleton was visualized with rhodamine-phalloidin staining, and bacterial strain 3152/19 was fluorescently
labeled with FAM-SE. Images represent the xy from z-stacks at 0.5-m m intervals.

DISCUSSION
One of the main challenges at present is how to treat bacterial biofilms, when anti-
biotic therapy alone is futile, especially if the biofilms are formed by antibiotic-resistant
strains. Moreover, the use of antibiotic therapy alone can complicate the outcome of

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FIG 6 Effect of the combination of NAC and CDN in a mouse pneumonia model. Mice were infected
with MDR strain 3152/19 and then treated (1 dose every 12 h) with humanized doses of NAC, CDN,
and a combination of CDN plus NAC. Results are expressed as bacterial counts obtained from the
lungs. Standard deviation bars are shown, and asterisks mark results that are statistically significant
(two-tailed Student’s t test: *, P , 0.05; **, P , 0.01; ***, P , 0.001).

the infection process, as it imposes a selective pressure that can end with the emer-
gence of new antibiotic-resistant strains (25, 27). In the search for antimicrobial alterna-
tives to overcome these recalcitrant infections, several authors have suggested combining
antibiotics with antibodies (24), antioxidants (25), or even phage lysins (28). In the case of
respiratory infections, patients suffering from COPD have recurrent pneumonia episodes
that, in many cases, are caused by the same strains that are colonizing their lower respira-
tory tract (29, 30). In these COPD patients, S. pneumoniae is one of the major etiologic
agents causing acute exacerbations (29, 30). Antibiotics are widely used in these patients,
and therefore, the risk of developing antibiotic resistance is an additional complication (29,
30). In the current study, when we compared the activity of CDN and CFM, both third-gen-
eration oral cephalosporins that are frequently used against respiratory infections, we
observed that CDN was more effective than CFM. CDN showed a greater inhibition of the
total growth and biofilm formation of S. pneumoniae. This is consistent with recent reports
showing that CDN had higher activity against S. pneumoniae clinical isolates, with lower
MIC50 and MIC90 values than other third-generation cephalosporins, including CFM and
cefpodoxime (15, 16).
In this study, as a complementary treatment against pneumococcal biofilms, we used
the mucolytic agent NAC, which presents thiol-reactive groups (31). This compound not
only has antimicrobial properties but also can be used to treat a wide range of respiratory
diseases, such as COPD and asthma (32). Moreover, the antioxidant and anti-inflammatory
properties of this compound have been used to treat cytokine storm and respiratory dis-
tress in coronavirus disease 2019 (COVID-19) patients (31). The antimicrobial properties of
NAC could be attributed to the competence of cysteine uptake by bacteria, based on the
disulfide bond reduction of proteins (affecting bacterial adhesion and biofilm formation),
or perturbation of the redox intracellular equilibrium (33, 34). Additionally, it has been sug-
gested that NAC could act as a weak acid in the biofilm, acidifying the biofilm matrix and
bacterial cytoplasm (35). In this sense, we recently demonstrated that NAC showed antimi-
crobial activity against polymicrobial biofilms of S. pneumoniae and S. aureus that were sus-
ceptible and resistant to methicillin (36).
We examined the efficacy of administration of the third-generation oral cephalo-
sporins CDN and CFM and the combination of each with NAC for the treatment and
prevention of pneumococcal biofilms. Treatment with CDN in combination with NAC
reduced the growth and biofilm formation of different strains of S. pneumoniae to a
much higher degree than the mixture of CFM with NAC or treatment with the cephalo-
sporins individually. This is consistent with previous studies showing that NAC has an
excellent safety and efficacy profile when used as an adjuvant molecule in the treat-
ment of bacterial biofilms (33, 37–39). The administration of NAC in association with

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tigecycline has been suggested as an alternative therapy to treat catheter-associated

infections (i.e., urinary tract infections, device-related infections) (40). Moreover, in
combination with different antibiotics (vancomycin, rifampicin, ciprofloxacin, azithro-
mycin, and tobramycin), NAC significantly promoted inhibition and/or eradication of
bacterial biofilms (41–43). One aspect that reinforces the relevance of our findings is
that few studies to date have focused on the combined treatment of biofilm-associ-
ated respiratory tract infections. In particular, the effect of NAC and ciprofloxacin on 15
different strains of Pseudomonas aeruginosa isolated from patients with suppurative
otitis media (44) confirmed the lack of growth when NAC (5 mg/mL) was used for treat-
ment, either alone or in combination with ciprofloxacin (44). Another study using 11 S.
pneumoniae strains showed that NAC alone had little activity against the planktonic
and sessile cultures, but when it was combined with three different antibiotics (amoxi-
cillin, erythromycin, and levofloxacin), a slightly enhanced activity against biofilms was
observed in some of the strains (45).
One aspect that reinforces the relevance of our findings is that few studies to date
have focused on the combined treatment of biofilm-associated respiratory tract infec-
tions. Moreover, there are no previous reports in the literature studying the combina-
tion of NAC with oral cephalosporins to prevent and treat S. pneumoniae biofilms
affecting the respiratory tract. In this sense, we have demonstrated the synergistic
effect of CDN and NAC to reduce S. pneumoniae adhesion to the lung epithelium using
A549 cells and for the treatment of acute pneumonia in a mouse model of infection.
Hence, our results are the first studies demonstrating the synergistic effect between
oral cephalosporins and antioxidants (here, CDN and NAC) for avoiding pneumococcal
adhesion in the lower respiratory tract and treating pneumococcal pneumonia. The in
vivo antimicrobial activity of both compounds was established using an MDR serotype
19A clinical isolate, as this serotype is prevalent in relapses and reinfections in COPD
patients associated with biofilms of the lower respiratory tract (46, 47). This is largely
relevant for patients suffering from chronic respiratory infections such as COPD, who
can develop recurrent and persistent infections by S. pneumoniae and other respiratory
pathogens (29, 30). One of the main problems with COPD patients is the lack of com-
plete clearance of the infection from the lower respiratory tract (48, 49), causing recur-
rent infections. Our findings promote the potential of CDN and NAC not only to reduce
S. pneumoniae adhesion to the lung epithelium but also to treat acute pneumonia epi-
sodes in which biofilms are already established. The use of NAC as an adjunctive ther-
apy to treat S. pneumoniae meningitis in combination with ceftriaxone has been tested
in a mouse meningitis model, although they did not find any therapeutic benefit (50).
However, our findings suggest that the combination of CDN and NAC could be useful
for treating respiratory infections associated with biofilms, as we found a significant
reduction in pneumococcal biofilms, including prevention of their formation and clear-
ance once established, and a decrease in the bacterial load in the lung compared to
the individual treatments.
Overall, our study demonstrates that the combination of CDN with NAC can prevent
and treat S. pneumoniae biofilms and reduce the adhesion of S. pneumoniae to the
lung epithelium, thus representing a promising treatment against pneumococcal
pneumonia. These results encourage a more widespread clinical use of NAC as an adju-
vant to different antibiotics, such as CDN, especially in microbial infections followed by
biofilm settlement (pneumonia, COPD exacerbations, otitis media, sinusitis), therefore
increasing the potential application of the combination of CDN and NAC as a preven-
tive and therapeutic treatment for microorganisms producing bacterial biofilms.

MATERIALS AND METHODS

Strains, media, and culture conditions. Table 1 shows the pneumococcal strains examined in this
study. We used clinical isolates of S. pneumoniae from our collection at the Spanish Pneumococcal
Reference Laboratory (SPRL) (National Center for Microbiology, Instituto de Salud Carlos III [ISCIII]). All
strains were from serotype 19A because it is a prevalent serotype frequently associated with multidrug
resistance, including to b -lactams, and because biofilm formation is markedly high in this particular
serotype (12, 51). The pneumococcal strains were cultured on Mueller-Hinton blood agar plates and

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Cefditoren and N-acetyl-L-Cysteine to Treat Biofilms Microbiology Spectrum

TABLE 1 Strains used in this study and MIC values for the different antibiotics and NACa
MIC (mg/mL) MIC (mg/mL)

Strain Serotype (source, description) Source PEN AMX CFM CDN CTX TET CHL ERY LVX NAC
3064/19 19A (pediatric/blood; bacteremic pneumonia) SPLR 0.12 0.12 2 0.12 0.25 0.5 4 0.25 1 2.5
1171/19 19A (pediatric/cerebrospinal fluid; meningitis) SPLR 2 2 >16 0.5 1 32 2 >128 1 2.5
3152/19 19A (pediatric/blood; bacteremia without focus) SPLR 4 8 >16 1 4 32 4 >128 1 2.5
aSPRL,Spanish Pneumococcal Reference Laboratory; PEN, penicillin; AMX, amoxicillin; CFM, cefixime; CDN, cefditoren; CTX, cefotaxime; TET, tetracycline; CHL,
chloramphenicol; ERY, erythromycin; LVX, levofloxacin; NAC, N-acetyl-L-cysteine.

incubated at 37°C and 5% CO2. Moreover, for liquid medium culture, all strains were grown in CpH8 me-
dium supplemented with 0.08% yeast extract (C1Y medium). Growth was controlled by measuring the
optical density at 550 nm (OD550).
Susceptibility testing and growth curves. The susceptibility of the different S. pneumoniae strains
of serotype 19A growing as planktonic cultures to the different b -lactams and the antioxidant NAC was
determined using the broth microdilution method, following the latest CLSI guidelines (52). The MICs of
penicillin (PEN), amoxicillin (AMX), CFM, CDN, and other antibiotics for the different strains are shown in
Table 1. The MIC of the antioxidant NAC is also shown in Table 1. CDN (oral cephalosporin) does not
have breakpoints following the EUCAST criteria. The strains in this study would be susceptible or susceptible
with increased exposure (intermediate) using the breakpoints for cefotaxime (parenteral cephalosporin) or
susceptible, intermediate, and resistant if the breakpoints for cefpodoxime (oral cephalosporin) were
considered.
For the activity assays on growing cells (growth curves), pneumococcal cells were grown into an
early stationary-phase culture in C1Y medium (OD550  0.5 to 0.6) and then diluted 100-fold into fresh
C1Y medium. Aliquots (200 m L per well) (4.5  106 CFU/mL) were added to a 96-well polystyrene micro-
titer plate (Falcon 3095; Corning). Different concentrations of CFM, CDN, and NAC and combinations of
an antibiotic plus the antioxidant were added at the beginning and then plates were incubated at 37°C
in an Epoch 2 device (BioTek Instruments), which acted as an incubator and read the OD550 value every
half hour.
Biofilm formation assay and antibiofilm therapy. Biofilm formation by pneumococcal cells of sero-
type 19A was induced using treated 96-well polystyrene microtiter plates (Costar 3595; Corning) as pre-
viously described (53). Briefly, cells were grown in C1Y medium up to the early stationary phase
(OD550  0.5 to 0.6), sedimented by centrifugation, resuspended in an equal volume of prewarmed C1Y
medium, diluted 100-fold, and then dispensed at a concentration of 200 m L per well (4.5  106 CFU/mL).
For the prevention/inhibition assays, different concentrations of CDN and NAC and combinations of
the two were added to the bacteria at the start of incubation on the plates, followed by 5 h of incuba-
tion at 37°C. Analysis of the effect was explored by crystal violet (CV) staining and viable cell counting,
as previously described (6, 10). For CV staining, after the 5-h incubation, total growth (A595) was meas-
ured using the BioTek Epoch 2 reader and then CV (0.2%) was added to stain the biofilm, which was
then incubated at room temperature for 15 min, followed by three washes with distilled water. After sol-
ubilization with 95% ethanol, the biofilm biomass (A595) was quantified in the reader. For viability (CFU
per milliliter), after the 5-h incubation, the planktonic culture was separated, washed with phosphate-
buffered saline (PBS), and gently disaggregated using a pipette. Tenfold serial dilutions were prepared
in PBS and then plated in the mentioned blood agar plates and incubated overnight at 37°C and 5%
CO2. Viable cells were quantified and expressed as CFU per milliliter.
For the treatment/disaggregation assays, different concentrations of CDN and NAC alone and com-
bined were added to the preformed biofilm after 4 h of incubation at 37°C. Analysis of the effect was
explored by viable cell counting. Briefly, after incubating the biofilm for 4 h at 37°C, the planktonic cul-
ture was separated, and the biofilm was washed with PBS. Then, the different concentrations of antibiot-
ics and antioxidants were added, and the biofilms were incubated for 1.5 h at 37°C, rinsed again with
PBS, and gently disaggregated using a pipette. Tenfold serial dilutions were then prepared in PBS. Viable
cells were quantified and expressed as CFU per milliliter.
Adhesion assays and confocal laser scanning microscopy. Experiments studying adhesion to
human epithelial cells were performed using A549 lung cells (CCL-185; ATCC), as previously described
(28). Monolayers of cells were cultured to 90 to 95% confluence in tissue culture flasks in RPMI 1640 me-
dium supplemented with 1% HEPES. We used 24-well treated plates (Falcon 353047; Corning) with 105
cells per well that were infected with 2  106 CFU/mL of strain 1171/19 or 3152/19, achieving a multi-
plicity of infection (MOI) of 10:1. Then, cells were incubated for 1 h at 37°C. After the initial incubation,
the supernatant was separated, the cells were washed three times with PBS, and CDN, NAC, or a combi-
nation was added; the cells were incubated for two additional hours at 37°C. After the final incubation,
the supernatant was separated, and the cells were washed again with PBS. To lift the infected A549 cells,
we first used a solution containing 0.25% trypsin-1 mM EDTA and then 0.025% saponin (in PBS), as previ-
ously described (28). From the mixture, 10-fold serial dilutions were prepared in PBS and plated onto
blood agar plates. The viable bacterial cells were quantified and expressed as CFU per milliliter.
For the CLSM assays, S. pneumoniae strains 1171/19 and 3152/19 were fluorescently labeled with
6-carboxyfluorescein-succinimidyl ester (FAM-SE; Molecular Probes), as previously described (54, 55).
A549 cells infected with FAM-SE-labeled bacteria were added to 12-mm circular coverslips for immuno-
fluorescence staining. We used a staining solution for 30 min at room temperature containing Hoechst

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Cefditoren and N-acetyl-L-Cysteine to Treat Biofilms Microbiology Spectrum

solution (Invitrogen) diluted 1/2,500 for DNA staining and rhodamine-phalloidin (Invitrogen) diluted
1/200 for actin cytoskeleton detection. We mounted the samples with Aqua-Poly/mount (Polysciences)
and observed them with the Leica spectral SP5 confocal microscope. The images represent the xy from
z-stacks at 0.5-m m intervals; the images were analyzed using the LAS AF software.
Pneumococcal pneumonia animal model. Experimental procedures involving mice were performed
at Instituto de Salud Carlos III (ISCIII), conforming to the Spanish government legislation (RD 53/2013, ECC/
566/2015) and European Community regulations (2010/63/EU). We used BALB/c male mice (8 to 12 weeks
old) weighing about 20 g that were bred by Charles River Laboratories. All animal procedures followed the
guidelines of the Bioethical and Animal Welfare Committee of ISCIII and Regional Authorities, which
reviewed and approved the protocols (PROEX 063.1/21). To study the treatment of pneumococcal pneu-
monia with CDN, NAC, or combinations, we used groups of five mice infected as previously described (55),
and we performed two independent experiments. To establish the animal model of pneumonia infection,
we chose the MDR strain 3152/19 (serotype 19A). Pneumococcal strains harboring antibiotic resistance,
such as those in serogroups 9, 14, 19, and 23, generally have low virulence in mice, and therefore, a high
dose has to be used (56, 57). Briefly, while the mice were under anesthesia with isoflurane, we inoculated
them intranasally with 50 m L of inoculum at a concentration of 109 CFU/mL (5  106 CFU per mouse); 24 h
after the challenge, the mice were treated with a placebo (PBS) or with humanized doses of CDN (5.7 mg/kg),
NAC (4.3 mg/kg), or the combination. We considered the standard human dose of 400 mg/12 h for CDN and
600 mg/day equivalent to 300 mg/12 h for NAC. Treatment was administered twice a day (every 12 h), follow-
ing the manufacturer’s indications. Finally, 48 h after the challenge, the mice were sacrificed by CO2, and bac-
terial counts were determined from samples recovered from the lung. The results were expressed as CFU per
milliliter of bacteria recovered from the lung.
Statistical analysis. Data presented in the manuscript represent results obtained from at least three
repeated independent experiments, representing at least three replicates. Statistical analyses were per-
formed using GraphPad InStat v. 8.0 (GraphPad Software). For comparison of two groups, we used the
two-tailed Student’s t test; for multiple comparisons, we used the analysis of variance (ANOVA) test, fol-
lowed by a post hoc test. Differences were considered statistically significant at P , 0.05 and highly sig-
nificant at P , 0.01 and P , 0.001.

ACKNOWLEDGMENTS
This work was supported by Ministerio de Ciencia e Innovación (MICINN) (grant
PID2020-119298RB-I00) and by Meiji Pharma Spain (grant MVP 119/20).
J.Y. has received grants from MSD-USA (MISP Call) and Pfizer that are not related to
this work. J.Y. has participated in advisory boards organized by MSD and Pfizer. M.G.
and P.C. are members of the Scientific Department of Meiji Pharma Spain. The other
authors declare no competing interests.

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